The Role Of MicroRNA-181a In Pre-eclampsia Through Regulating Proliferation And Immunosuppressive Properties In Mesenchymal Stem Cells

Mesenchymal stem cells (MSC) not only possess extensive self-renewal potential and the ability to modulate immunocyte activation, but they are also easily expanded and stored ex vivo, and are considered to be "immunoprivileged". Under stress conditions, MSC home to sites of injury whereby they decrease local inflammation and participate to tissue repair and regeneration by secretion of cytokines, chemokines and extracellular matrix proteins. MSC modulate the function of immune cells including T and B lymphocytes, dendritic cells, and natural killer cells, inhibit their proliferation and alter their cytokines secretion. The first successful isolation of fibroblast-like colonies of MSC is from bone marrow. Maternal-fetal interface is also an important source of MSC, several groups have isolated MSC from umbilical cord, placenta and decidua. However, it is reported that MSC separation using traditional cell adherent method can’t have a good results since the MSC is rare in tissue. Florian et al. also reported that cell isolation protocols have a major impact on the functional activity of bone marrow-derived progenitor cells which show the importance of MSC isolation protocols optimization.It should be noted that all the method used to isolate MSC required to digest the tissue to obtain a single cell suspension. Since umbilical cords is hard to digested by traditional digestive enzymes collagen, and long term digestion will affect the cell vitality, there is a dilemma between obtaining a single cell suspension and cell vitality. Therefore, we combine the method of isolate MSC from plastic-adherent tissue and the method of isolate MSC from digested single cell suspension, use a semi-tissue-adherent method successfully to quickly obtain large amount of UCMSC (Umbilical cords mesenchymal stem cells). It seems that a mild digestion of umbilical cords will release some MSC in the tissue, and it also will make the not complete digested tissue easier to adhere to plastic, and the MSC inside the tissue will migrate to the plastic and expansion. We also improved the formula of digestive enzymes, add hyaluronidase and dispase enzyme which will prevent unwanted clumping of cells cultured in suspension.The results shows by using our method of isolate MSC, adherent cells with fibroblastic morphology could be observed as early as24hours. The cells formed a monolayer of homogenous bipolar spindle-like cells with a whirl pool like array within1week. Then, UCMSC were serially passaged to examine their expansion potential and determined to expand readily for up to2weeks. After3cell passages, the adherent cells were symmetric with phenotypic surface antigens. The UCMSC shared most of their immunophenotype with bone marrow-derived MSC (BMMSC) as reported, including positivity for CD29, CD44, CD90, CD105(SH2), CD73(SH3) and HLA-ABC, negativity for CD19, CD11b, CD14, CD34and CD31(endothelial cell marker) and HLA-DR.In order to determine whether the UCMSC also had immunosuppressive effects on the proliferation of T lymphocytes in response to mitogenic stimulation in vitro, purified T cells were stimulated with Phytohaemagglutinin (PHA) in the presence or absence of UCMSC. After72hours, T cells were harvested, and T-cell proliferation was measured using [3H] thymidine incorporation. The results demonstrated that after72hours coculture, the UCMSC inhibited the proliferation of T cell in response to mitogen treatment. We next investigated whether T cells activation markers expression was interfered by UCMSC. T cells were harvested after24hours coculture and CD8+cells were analyzed for the expression of CD25, CD69and intracellular IFN-y production. After T cells were in contact with UCMSC, IFN-y production was impaired, and the expression of CD25and CD69was also a slightly affected.To determine whether UCMSC secreted several soluble factors to exercise its immunosuppressive properties, we measured mRNA for IL-6, TGFB, IDO, VEGF and COX-2in UCMSC, and the protein level of IL-6, TGF-β, VEGF and COX-2was also detected by ELISA or western blot. UCMSC secreted significant amounts of VEGF, IL6and COX-2, but they produced only minimal amounts of TGF-β. In addition, the basal expression of IDO in UCMSC is very low, but a substantial amount mRNA and protein of IDO can be induced by IFN-y. To further investigate whether those soluble factors interfere with the immunosuppressive results of MSC, anti-IL-6and anti-TGF-β neutralizing Ab,1-M-Trp, an inhibitor of IDO enzymatic activity, and indomethacin, an inhibitor of PGE2enzymatic activity were added to the cultures, respectively. Our results showed that pretreatment with1-MT significantly reversed UCMSC mediated inhibition of T cells proliferation. However, treatment with neutralizing Ab for TGF-β1and IL-6, inhibitor of PGE2could not reverse the inhibitory effects of UCMSC on T cells proliferation. These results suggest that IDO, not IL-6, TGF-β or PGE2, may contribute, in part, to UCMSC-mediated suppression of T cells.In order to go deep into the function of MSC in the maternal-fetal interface, pre-eclampsia was chose as a disease model to investigate MSC. Pre-eclampsia (PE) is a disease characterized by hypertension and proteinuria during pregnancy (appeared after20weeks of gestation). Its pathogenesis is not completely understood. Previous studies indicate that PE may be a pregnancy-induced autoimmune disease and the unbalanced immune system in the maternal-fetal interface may be one cause of PE. In human pregnancy, there is a shift of cytokine production toward Th2-type immunity with predominance of IL-4and IL-10over IL-2and TNF-a in stimulated peripheral blood mononuclear cells (PBMC). Thus, pregnancy is considered a Th2phenomenon, while Thl cytokines increase and Th2cytokines decrease in PBMC from women with PE, implying the balance shifts toward Thl immunity in PE. In additional, our previous date indicates that in preeclamptic patients, decreased percentages of Tc2and Th2cells and the increased ratios of Tcl/Tc2can be detected in both decidua and maternal peripheral blood. Specifically, one of the increased Thl-type cytokines TNF-a is associated with PE and has been speculated to contribute to the disease. Autoantibody-mediated angiotensin receptor activation contributes to preeclampsia through TNF-a. Moreover, angiotensin receptor autoantibody and TNF-α both affect the production of soluble fms-like tyrosine-1and soluble endoglin which are two critical etiological factor in pre-eclampsia.Several strategies have been developed targeting specific factors relevant to PE pathogenesis. However, none of the therapy was focused on the immune disorder in PE, it is urgent to develop more effective therapy for PE based on target the disorder immune system in PE., we develop a Thl cell-induced PE mice model as reported, and targeting TNF-a which have both an important role in this PE model pathogenesis and human PE pathogenesis to develop a novel effective therapy.Other and our previous data demonstrated the immunosuppressive and anti-inflammatory effects of MSC in the treatment of several animal disease models including autoimmune diseases. As a result of these unique qualities, MSC are attractive candidates in stem cell-based strategies for PE.Therefore, in this study, we developed a Thl cell-induced PE mice model as reported, and target the pathogenesis factor TNF-a using systemic infusion of UCMSC. Our results showed that systemic infusion with UCMSC protected mice against Th1-induced PE, which revealed here as a decrease in blood pressure, proteinuria and fetal rejection rate (compared with Thl cells adoptively transferred mouse at day14). In addition, MSC infusion also have beneficial effects on feto-placental development including increased placental weights, fetal weights and fetal numbers compared with Thl cells adoptively transferred mouse. Histologically, MSC infusion also reversed the condition of PE in kidney and placenta. These findings suggest that cell-based therapy using UCMSC can ameliorates Thl-induced PE in mice. More importantly, systemic infusion with UCMSC significantly attenuated the TNF-a expression in uterine and spleen lymphocytes and slightly attenuated the IFN-y and IL-4expression in spleen lymphocytes.These compelling findings suggest that UCMSC reverse the cytokine abnormal in PE uterine and spleen lymphocytes.We first reported UCMSC-based therapy ameliorated both clinical and histopathological severity of PE symptoms, decreased the blood pressure and proteinuria, suppressed glomerulonephritis, protected the feto-placental development and reversed the TNF-a abnormal expression in uterine and spleen lymphocytes.The results also remind us the dysfunction of MSC in the maternal-fetal interface might be a new mechanism involving the pathogenesis of PE.However, the mechanism involving dysfunction of MSC reminds unkonwn.MiRNAs have emerged as a new dimension of gene regulation in recent years. They are short(19-25nucleotides), single-stranded and non-protein-coding RNAs that regulate gene expression by binding to the3’untranslated region (3’UTR) of the target mRNAs. They function in diverse biological processes, including development, differentiation, apoptosis and oncogenesis. We previous reported that miR-181a were significantly increased in placenta from women with severe PE. miR-181a is reported can suppress cell proliferation in cancer cells and regulate immunocyte activation. However, the exact function of miR-181a in MSC was not been well characterized.In this study, we aimed to investigate if miR-181a regulate MSC proliferation and immunosuppressive properties through modulation several important pathways. We found the basal expression of miR-181a in the MSC derived from the umbilical cord and decidua of PE patients increased. Transfection of miR-181a oligo prevented the proliferation of MSC, but did not affect MSC apoptosis. The overexpression of miR-181a prevented the activating of TGF-β signaling pathway and down-regulated the mRNA and protein levels of the miR-181a targeting gene TGFBR1and TGFBRAP1, suggesting that miR-181a acts as an endogenous attenuator of TGF-P signaling pathway in MSC. Putative miR-181a binding sites down-regulated the TGFBR1and TGFBRAP13’-UTR reporter activity, suggesting that miR-181a bind to TGFBR1and TGFBRAP13’-UTR. On the other hand, transfection of miR-181a oligo enhanced IL6and IDO expression in MSC by activing p38and JNK signaling pathway individually. miR-181a transfected MSC even enhanced the proliferation of T cells in a short term culture, suggesting that miR-181a attenuates immunosuppressive properties of MSC in vitro. Together, our data demonstrated that miR-181a as a regulator of both proliferation and immunosuppressive properties in MSC. miR-181a act as important endogenous negative regulators of TGFBR1and TGFBRAP1, which inhibit TGF-β signaling pathway and prevent the proliferation of MSC. miR-181a also regulates immunosuppressive properties of MSC through MAPK signaling pathway, enhanced IL6and IDO expression and this resulted in the ability of miR-181a to attenuates immunosuppressive properties of MSC in vitro.We next determined the in vivo effects of miR-181a to MSC-based therapy. The immunosuppressive and anti-inflammatory effects of MSC have been demonstrated in the treatment of several animal disease models, including graft-versus host disease, diabetes, rheumatoid arthritis, autoimmune encephalomyelitis, systemic lupus erythematosus, periodontitis, inflammatory bowel disease (IBD) and sepsis. Our study proved that systemic infusion with MSC have beneficial effects on healing a model of pre-eclampsia in mouse induced by adoptive transfering of activated Th1cells. Although Thl cells induced pre-eclampsia models have features of preeclampsia, this model is still poor overall models of the human disease and is not a widly used mouse model, because they do not include the complete spectrum of pathophysiological changes associated with preeclampsia. Therefore, PE animal model was not used to investigate the immunosuppressive and repair capacity of MSC in vivo.Recently, several groups have reported that treatment with human MSC exhibits early efficacy in attenuating the progression of experimental colitis induced by dextran sulfate sodium (DSS) which is a widly used mouse model to evaluate the immune and inflammatory status. Similar to previous reports, we confirmed that oral administration of3%DSS for8days induced acute colitis in C57BL/6mice characterized by an overall elevation of colitis scores based on the presence of sustained weight loss, bloody diarrhea/loose feces. DSS treated mice also shows shorter colons, lighter colons and higher mortality. Histological studies revealed severe colonic transmural inflammation with increased wall thickness and localized inflammatory cell infiltration, epithelial ulceration with degeneration of crypt architecture and loss of goblet cells.Systemic infusion with MSC protected mice against colitis-related tissue injuries and reduced the overall disease severity, shown here as a decrease in disease score, reversing and stabilizing of body weight compared with treatment with DSS alone at day11, suppressing of colonic inflammation. MSC treated mouse had longer length of colons, heavier weight of colons and lower mortality compared with DSS treated mouse on day12. Histologically, MSC significantly ameliorated colonic transmural inflammation and decreased wall thickness, restored goblet cells and suppressed mucosal ulceration and focal loss of crypts, thus restoring normal intestinal architecture.Importantly, our results showed that miR-181a attenuates the MSC-based protection to DSS treated mouse against colitis-related tissue injuries. The overall disease scores has no significantly difference compared with DSS treated mouse. Systemic infusion with miR-181a transfected MSC neither reverse or stabilize body weight, nor suppress colonic inflammation. Compared with the MSC treated mice where the gross appearance of colons remain normal, miR-181a transfected MSC treated mouse also showed typical signs of severe acute inflammation. Length of colons in the miR-181a transfected MSC treated mouse was significantly shortened and the weight of colons in the miR-181a transfected MSC treated mouse was significantly lighter compared with the MSC treated mice. Histologically, systemic infusion with miR-181a transfected MSC shows no difference compared with DSS treated mice.